HIC Shows Promising Separation of Deaminated and Non-Deaminated AAV Capsids

A team at Oxford Biomedica conducted studies that showed the effectiveness of using HIC to separate non-deamidated capsids from deamidated capsids.

During a presentation at the American Society of Gene and Cell Therapy’s (ASGCT’s) 27th Annual Meeting, Alex Meola, associate director of AAV Downstream Process Development at Oxford Biomedica, presented findings from a study conducted by him and his team. The study showcased the effectiveness of hydrophobic interaction chromatography (HIC) in separating non-deamidated capsids from deamidated capsids. The ASGCT annual meeting will take place from May 7–11, 2024 in Baltimore, Md.

Meola highlighted the increasing importance of studying the post-translational modification of capsid proteins of the adeno-associated virus (AAV) in the field of gene therapies. The industry has been giving special attention to the connection between the deamidation of capsid protein from viral protein 1 (VP1)-specific residues, such as the N57 domain, and a decrease in in-vivo potency.

According to Meola, certain factors such as temperature, pH, and storage time have been found to be significant contributors to this phenomenon. At Oxford Biomedica, we have developed a straightforward screening protocol to address this issue. We examine a range of residents to determine their static binding capacity. For this particular situation, we chose to examine individuals who are able to tolerate high levels of salt," he explained.

In addition to implementing process control measures to prevent deamidation, Meola's team theorized that the extrusion of VP1 and the deamidation of N57 are interconnected processes that lead to the formation of empty, partial, and full capsids. These variations in capsid formation have a significant impact on the effectiveness of the anion exchange chromatography (AEX) process. The effectiveness of AEX in removing empty capsids and delivering functional AAV is therefore compromised.

According to Meola, the current charge-driven separation techniques do not offer enough resolution to distinguish between deamidated and non-deamidated capsid species. As a result, his team emphasized innovative methods that can be used in the AEX process to tackle the difficult task of eliminating both empty capsids and deamidated intact capsids. "It is important to study and define controlling strategies in the upstream and downstream processes early on in process development. This will help limit the onset of deamidation, minimize product-related impurities, and prevent loss of vector functionality," Meola explained.

One of the team's initial strategies involved utilizing HIC to effectively distinguish peaks with almost no overlap. Following separation, every peak was isolated and processed separately using AEX. It was surprising to find that HIC was able to separate two distinct species of AAV capsids with almost perfect resolution. Every species underwent reprocessing on AEX, and the quality attributes of the resulting intermediate peaks were evaluated. According to his explanation, it was observed that the capsids with higher hydrophobicity also had a greater negative charge.

In addition, the data obtained from liquid chromatography-mass spectrometry analysis showed that capsids with higher hydrophobicity and more negative charge had noticeable levels of VP1-specific N57 deamidation. This deamidation has been associated with a decrease in gene expression. It was shown that the extrusion of VP1 reveals the hydrophobic phospholipase A2 domain on the VP1 unique region. This exposes the VP1 N57 residue to the solvent, making it susceptible to deamidation.

The team successfully showed that HIC is an effective technique for distinguishing between non-deamidated capsids and deamidated capsids. Meola also mentioned that the team is developing a unique method to remove deamidated species and enhance full capsids during the AEX process.

Source: annualmeeting.asgct.org; PubMed